Synergistic Effect of H2O2 and NO2 in Cell Death Induced by Cold Atmospheric He Plasma (original) (raw)

Cold atmospheric pressure plasmas (CAPPs) have emerged over the last decade as a new promising therapy to fight cancer. CAPPs' antitumor activity is primarily due to the delivery of reactive oxygen and nitrogen species (RONS), but the precise determination of the constituents linked to this anticancer process remains to be done. In the present study, using a micro-plasma jet produced in helium (He), we demonstrate that the concentration of H 2 O 2 , NO 2 − and NO 3 − can fully account for the majority of RONS produced in plasma-activated buffer. The role of these species on the viability of normal and tumour cell lines was investigated. Although the degree of sensitivity to H 2 O 2 is cell-type dependent, we show that H 2 O 2 alone cannot account for the toxicity of He plasma. Indeed, NO 2 − , but not NO 3 − , acts in synergy with H 2 O 2 to enhance cell death in normal and tumour cell lines to a level similar to that observed after plasma treatment. Our findings suggest that the efficiency of plasma treatment strongly depends on the combination of H 2 O 2 and NO 2 − in determined concentrations. We also show that the interaction of the He plasma jet with the ambient air is required to generate NO 2 − and NO 3 − in solution. Cancer is a leading cause of death worldwide and its incidence rate increases with the age of the population, the exposure to carcinogens and the modern lifestyle of the population. About two thirds of patients defeat their disease, and the combined action of surgery, radiotherapy and chemotherapy accounts for most cured cases 1. Alongside with these classical therapies, new therapies have emerged, such as anti-angiogenic therapy and immunotherapy 1. However, therapy resistance has been observed with every type of therapy that is available today, including poly-chemotherapy, radiotherapy, immunotherapy, and molecular targeted therapy 2. Importantly, sequencing of primary tumors has revealed that therapy-resistant clones already exist prior to targeted therapy, demonstrating that tumor heterogeneity in primary tumors confers a mechanism for inherent therapy resistance 2. Therefore, there is still the need of a new therapy that can overcome this problem. There are numerous publications showing that cold atmospheric pressure plasmas (CAPPs) are effective against tumour cells both in vitro and in vivo (ref. 3 and references therein). CAPPs are partially ionised gases containing a complex and reactive environment consisting of ions, electrons, free radicals, strong localised electric field, UV radiation, and neutral molecules. CAPPs' devices are classified in three categories: direct plasma sources that use the target as a counter electrode [e.g. floating electrode dielectric barrier discharge (FE-DBD)]; indirect plasma sources that do not use the target as a counter electrode (e.g. plasma jets); and hybrid plasma sources that combine the benefits of direct and indirect plasma sources 4-10. Different gases can be used to produce CAPPs such as Helium (He), Argon (Ar), Nitrogen (N 2), ambient air, or a mixture of gases 6,7. All the plasma sources developed for biomedical applications have in common that the major reactive molecules produced in CAPPs emerge when the components of the partially ionized gas (atoms, molecules, ions and electrons) interact with the molecules of the surrounding air, i.e. O 2 , N 2 and H 2 O, and with the biological sample which is usually a wet surface (e.g. cells in medium) 11-14. Consequently, the plasma composition and the subsequent effects on cells can vary enormously depending on the plasma source, the plasma settings, the ambient conditions and the biological target 12,15. Despite this large variability in the plasma composition, it is now widely accepted that the principal mode of plasma-cell interaction is the delivery of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that